Organ function relies upon the appropriate attributes of each of its individual operational components. For example, in the embryonic vertebrate heart, effective propulsion of circulation depends upon the distinct morphological, electrophysiological, and contractile traits of the atrial and ventricular chambers. Despite centuries of awareness of the key differences between atrial and ventricular cardiomyocytes, the fundamental mechanisms that allocate cells into chamber-specific lineages and direct chamber-specific differentiation remain largely mysterious. Our laboratory's research focuses on understanding the genetic pathways responsible for chamber fate assignment. By exploiting the utility of the zebrafish as a model organism, we have shown that the BMP and FGF signaling pathways differentially affect atrial and ventricular cell numbers, providing important clues to the mechanisms that initially establish the atrial and ventricular progenitor pools. Furthermore, our preliminary data indicate that initial chamber fate decisions can be plastic and that mechanisms exist to maintain chamber- specific characteristics in differentiated myocardium. Together, our studies suggest an intriguing model in which early patterning of the heart field, followed by later reinforcement of chamber identity, results in proper chamber fate assignment. Here, we propose to evaluate aspects of this model in detail. In Aim 1, we will delve deeper into the mechanisms responsible for the initial establishment of chamber progenitor pools. Specifically, we will use fate mapping, time-lapse tracking, mosaic analysis, and evaluation of candidate effector genes to determine how BMP signaling promotes the establishment of atrial progenitor cells. In Aim 2, we will investigate the mechanisms that maintain chamber identity. Employing transgenic reporters of chamber identity, time-lapse analysis, mosaic analysis, and dissection of chamber-specific regulatory sequences, we will test whether FGF signaling functions to insure maintenance of ventricular chamber identity by promoting expression of nkx genes. Finally, in Aim 3, we will pursue identification of new pathways that regulate chamber fate assignment, taking advantage of our discovery of 4 intriguing compounds that impact atrial or ventricular cardiomyocyte production. Together, our studies will illuminate new features of the network of pathways controlling chamber fate assignment. In the long term, this work has the potential to shed light on the causes of cardiac birth defects and to facilitate innovations in regenerative medicine.